D-polyglutamine amyloid recruits L-polyglutamine monomers and kills cells.
ABSTRACT: Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a "rippled ?-sheet" interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.
Project description:Polyglutamine (PolyQ) aggregates are a hallmark of several severe neurodegenerative diseases, expanded CAG-repeat diseases in which inheritance of an expanded polyQ sequence above a pathological threshold is associated with a high risk of disease. Application of vibrational circular dichroism (VCD) reveals that these PolyQ fibril aggregates exhibit a chiral supramolecular organization that is distinct from the supramolecular organization of previously observed amyloid fibrils. PolyQ fibrils grown from monomers with Q repeats 35 and above (Q?35) exhibit approximately 10-fold enhancement of the same VCD spectrum compared to the already enhanced VCD of fibrils formed from Q repeats 30 and below (Q?30).
Project description:Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K2Q24K2W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both (12)C and (13)C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a ?-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single ?-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.
Project description:Amyloid aggregates of Tau protein have been implicated in etiology of many neurodegenerative disorders including Alzheimer's disease (AD). When amyloid growth is induced by seeding with preformed fibrils assembled from the same protein, structural characteristics of the seed are usually imprinted in daughter generations of fibrils. This so-called conformational memory effect may be compromised when the seeding involves proteins with non-identical sequences leading to the emergence of distinct structural variants of fibrils (amyloid 'strains'). Here, we investigate cross-seeding of full-length human Tau (FL Tau) with fibrils assembled from K18 and K18?K280 fragments of Tau in the presence of poly-L-glutamate (poly-Glu) as an enhancer of Tau aggregation. To study cross-seeding between Tau polypeptides and the role of the conformational memory effect in induction of Tau amyloid polymorphism, kinetic assays, transmission electron microscopy, infrared spectroscopy and limited proteolysis have been employed. The fastest fibrillization was observed for FL Tau monomers seeded with preformed K18 amyloid yielding daughter fibrils with unique trypsin digestion patterns. Morphological features of daughter FL Tau fibrils induced by K18 and K18?K280 seeds were reminiscent of the mother fibrils (i.e. straight paired fibrils and paired helical filaments (PHFs), respectively) but disappeared in the following generations which became similar to unpaired FL Tau amyloid fibrils formed de novo. The structural evolution observed in our study was accompanied by disappearance of the unique proteolysis profile originated from K18. Our findings may have implications for understanding molecular mechanisms of the emergence and stability of Tau amyloid strains.
Project description:The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
Project description:Amyloid fibrils are cross-?-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-?-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.
Project description:Amyloid fibrils generally display chirality, a feature which has rarely been exploited in the development of therapeutics against amyloid diseases. This study reports, for the first time, the use of mesoscopic chiral silica nanoribbons against the in vivo amyloidogenesis of human islet amyloid polypeptide (IAPP), the peptide whose aggregation is implicated in type 2 diabetes. The thioflavin T assay and transmission electron microscopy show accelerated IAPP fibrillization through elimination of the nucleation phase and shortening of the elongation phase by the nanostructures. Coarse-grained simulations offer complementary molecular insights into the acceleration of amyloid aggregation through their nonspecific binding and directional seeding with the nanostructures. This accelerated IAPP fibrillization translates to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental, and behavioral assays. This study has implicated the potential of employing chiral nanotechnologies against the mesoscopic enantioselectivity of amyloid proteins and their associated diseases.
Project description:In Huntington's disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A "protective" C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (htt(NT)) adopts an ?-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the ?-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt ?-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the htt(NT) ?-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like ?-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.
Project description:Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) involve the abnormal accumulation in the brain of filaments composed of both three-repeat (3R) and four-repeat (4R) (3R/4R) tau isoforms. To probe the molecular basis for AD's tau filament propagation and to improve detection of tau aggregates as potential biomarkers, we have exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking-induced conversion or AD RT-QuIC). The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils made of certain recombinant tau fragments. AD RT-QuIC detected seeding activity in AD (n = 16) brains at dilutions as extreme as 107-1010-fold, but was 102-106-fold less responsive when seeded with brain from most cases of other types of tauopathy with comparable loads of predominant 3R or 4R tau aggregates. For example, AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to the weakest of the AD specimens, and higher than 3 of 4 specimens with 3R/4R primary age-related tauopathy. AD seeds shared properties with the tau filaments found in AD brains, as AD seeds were sarkosyl-insoluble, protease resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity exhibited by AD and CTE tau filaments compared to other types of tauopathies in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides rapid ultrasensitive quantitation of 3R/4R tau-seeding activity as a biomarker.
Project description:Lipid membranes are suggested as the primary target of amyloid aggregates. We study aggregates formed by a polyglutamine (polyQ) peptide, and their disruptive effect on lipid membranes. Using solution atomic force microscopy (AFM), we observe polyQ oligomers coexisting with short fibrils, which have a twisted morphology that likely corresponds to two intertwined oligomer strings. Fourier transform infrared spectroscopy reveals that the content of ?-sheet enriched aggregates increases with incubation time. Using fluorescence microscopy, we find that exposure to polyQ aggregates results in deflated morphology of giant unilamellar vesicles. PolyQ aggregates induced membrane disruption is further substantiated by time-dependent calcein leakage from the interior to the exterior of lipid vesicles. Detailed structural and mechanical perturbations of lipid membranes are revealed by solution AFM. We find that membrane disruption by polyQ aggregates proceeds by a two-step process, involving partial and full disruption. In addition to height contrast, the resulting partially and fully disrupted bilayers have distinct rigidity and adhesion force properties compared to the intact bilayer. Specifically, the bilayer rigidity increases as the intact bilayer becomes partially and fully disrupted. Surprisingly, the adhesion force first decreases and then increases during the disruption process. By resolving individual fibrils deposited on bilayer surface, we show that both the length and the number of fibrils can increase with incubation time. Our results highlight that membrane disruption could be the molecular basis of polyQ aggregates induced cytotoxicity.
Project description:Amyloid-like fibrils formed by huntingtin exon-1 (htt_ex1) are a hallmark of Huntington's disease (HD). The structure of these fibrils is unknown, and determining their structure is an important step toward understanding the misfolding processes that cause HD. In HD, a polyglutamine (polyQ) domain in htt_ex1 is expanded to a degree that it gains the ability to form aggregates comprising the core of the resulting fibrils. Despite the simplicity of this polyQ sequence, the structure of htt_ex1 fibrils has been difficult to determine. This study provides a detailed structural investigation of fibrils formed by htt_ex1 using solid-state nuclear magnetic resonance (NMR) spectroscopy. We show that the polyQ domain of htt_ex1 forms the static amyloid core similar to polyQ model peptides. The Gln residues of this domain exist in two distinct conformations that are found in separate domains or monomers but are relatively close in space. The rest of htt_ex1 is relatively dynamic on an NMR time scale, especially the proline-rich C-terminus, which we found to be in a polyproline II helical and random coil conformation. We observed a similar dynamic C-terminus in a soluble form of htt_ex1, indicating that the conformation of this part of htt_ex1 is not changed upon its aggregation into an amyloid fibril. From these data, we propose a bottlebrush model for the fibrils formed by htt_ex1. In this model, the polyQ domains form the center and the proline-rich domains the bristles of the bottlebrush.